Publications 2012

Combined in vivo multiphoton and CARS imaging of healthy and disease-affected human skin

Breunig HG, Bückle R, Kellner-Höfer M, Weinigel M, Lademann J, Sterry W, König K.

Microsc Res Tech. 2012 Apr;75(4):492-8. doi: 10.1002/jemt.21082. Epub 2011 Oct 3.


We present combined epi-coherent anti-Stokes Raman scattering (CARS) and multiphoton imaging with both chemical discrimination and subcellular resolution on human skin in vivo. The combination of both image modalities enables label-free imaging of the autofluorescence of endogenous fluorophores by two-photon excited fluorescence, as well as imaging of the distribution of intercellular lipids, topically applied substances and water by CARS. As an example for medical imaging, we investigated healthy and psoriasis-affected human skin with both image modalities in vivo and found indications for different lipid distributions on the cellular level.


Nanosurgery of cells and chromosomes using near-infrared twelve-femtosecond laser pulses

Uchugonova A, Lessel M, Nietzsche S, Zeitz C, Jacobs K, Lemke C, König K.

J Biomed Opt. 2012 Oct;17(10):101502. doi: 10.1117/1.JBO.17.10.101502.


Laser-assisted surgery based on multiphoton absorption of near-infrared laser light has great potential for high precision surgery at various depths within the cells and tissues. Clinical applications include refractive surgery (fs-LASIK). The non-contact laser method also supports contamination-free cell nanosurgery. In this paper we describe usage of an ultrashort femtosecond laser scanning microscope for sub-100 nm surgery of human cells and metaphase chromosomes. A mode-locked 85 MHz Ti:Sapphire laser with an M-shaped ultrabroad band spectrum (maxima: 770  nm/830  nm) and an in situ pulse duration at the target ranging from 12 fs up to 3 ps was employed. The effects of laser nanoprocessing in cells and chromosomes have been quantified by atomic force microscopy. These studies demonstrate the potential of extreme ultrashort femtosecond laser pulses at low mean milliwatt powers for sub-100 nm surgery of cells and cellular organelles.


Safety assessment by multiphoton fluorescence/second harmonic generation/hyper-Rayleigh scattering tomography of ZnO nanoparticles used in cosmetic products

Darvin ME, König K, Kellner-Hoefer M, Breunig HG, Werncke W, Meinke MC, Patzelt A, Sterry W, Lademann J.

Skin Pharmacol Physiol. 2012;25(4):219-26. doi: 10.1159/000338976. Epub 2012 May 30.


Zinc oxide nanoparticles (ZnO NPs) are commonly used as UV filters in commercial sunscreen products. Their penetration into the skin is intensively discussed in the literature. In the present in vivo study, penetration of ZnO NPs (30 nm in size) into human skin was investigated by multiphoton tomography. Based on the non-linear effects of a second harmonic generation and hyper-Rayleigh scattering, the distribution of ZnO NPs in the horny layers of the epidermis, as well as the furrows, wrinkles and orifice of the hair follicles was analyzed. This method permitted distinguishing between the particulate and dissolved forms of Zn. A detection limit of 0.08 fg/μm(3) was estimated. Taking advantage of this sensitivity, it was clearly shown that ZnO NPs penetrate only into the outermost layers of stratum corneum, furrows and into the orifices of the hair follicles and do not reach the viable epidermis.


Multiphoton laser microscopy and fluorescence lifetime imaging for the evaluation of the skin

Seidenari S, Arginelli F, Bassoli S, Cautela J, French PM, Guanti M, Guardoli D, König K, Talbot C, Dunsby C.

Dermatol Res Pract. 2012;2012:810749. doi: 10.1155/2012/810749. Epub 2011 Nov 28.


Multiphoton laser microscopy is a new, non-invasive technique providing access to the skin at a cellular and subcellular level, which is based both on autofluorescence and fluorescence lifetime imaging. Whereas the former considers fluorescence intensity emitted by epidermal and dermal fluorophores and by the extra-cellular matrix, fluorescence lifetime imaging (FLIM), is generated by the fluorescence decay rate. This innovative technique can be applied to the study of living skin, cell cultures and ex vivo samples. Although still limited to the clinical research field, the development of multiphoton laser microscopy is thought to become suitable for a practical application in the next few years: in this paper, we performed an accurate review of the studies published so far, considering the possible fields of application of this imaging method and providing high quality images acquired in the Department of Dermatology of the University of Modena.


Hybrid multiphoton multimodal tomography of in vivo human skin

König K

Volume 1, Issue 1   July/August/September 2012 Pages 11 - 26


Clinical multiphoton tomography based on femtosecond near infrared laser pulses for in vivo high-resolution skin imaging has been employed to thousands of volunteers and patients. Two-photon cellular autofluorescence and second harmonic generation of collagen can be detected with single-photon sensitivity and submicron spatial resolution. Also in vivo clinical CARS has been realized to image intratissue lipids and water. Novel developments focus on multimodal hybrid imaging to generate optical tissue biopsies with subcellular resolution, deep-tissue information, and chemical fingerprints. Wide-field imaging tools such as dermoscopes, optical coherence tomographs as well as ultrasound and photoacoustic devices can be integrated. The hybrid tomographs have the potential to trace cosmetics and pharmaceutical components such as sunscreen nanoparticles and anti-aging products in humans. Skin cancer such as malignant melanoma and basal cell carcinoma as well as dermatitis can be detected at an early stage and the efficiency of the treatment can be monitored. These novel hybrid multimodal multiphoton tomographs may become important biopsy-free and label-free imaging tools in personalized medicine, pharmacy, biotechnology as well as cosmetic research.


Multiphoton imaging of freezing and heating effects in plant leaves

Breunig HG, Tümer F, König K.

J Biophotonics. 2012 Sep 14. doi: 10.1002/jbio.201200093.


Thermally-induced changes in Arabidopsis thaliana leaves were investigated with a novel cryo microscope by multiphoton, fluorescence lifetime and spectral imaging as well as micro spectroscopy. Samples were excited with fs pulses in the near-infrared range and cooled/heated in a cryogenic chamber. The results show morphological changes in the chloroplast distribution as well as a shift from chlorophyll to cell-wall fluorescence with decreasing temperature. At temperatures below -40 °C, also second harmonic generation was observed. The measurements illustrate the suitability of multiphoton imaging to investigate thermally-induced changes at temperatures used for cryopreservation as well as for basic investigations of thermal effects on plant tissue in general (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).


Cancer-cell killing by engineered Salmonella imaged by multiphoton tomography in live mice

Uchugonova A, Zhao M, Zhang Y, Weinigel M, König K, Hoffman RM.

Anticancer Res. 2012 Oct;32(10):4331-7.


Our laboratory has previously developed a bacterial cancer therapy strategy by targeting tumors using engineered Salmonella typhimurium auxotrophs (S. typhimurium A1-R) that were generated to grow in viable as well as necrotic areas of tumors but not in normal tissue. The mechanism by which A1-R kills cancer cells is unknown. In the present report, high-resolution multiphoton tomography was used to investigate the cellular basis of bacteria killing of cancer cells in live mice. Lewis lung cancer cells (LLC) were genetically labeled with red fluorescent protein (RFP) and injected subcutaneously in nude mice. After tumor growth was observed, the mice were treated with A1-R bacteria expressing GFP, via tail-vein injection. Mice without A1-R treatment served as untreated controls. The imaging system was 3D scan head mounted on a flexible mechano-optical articulated arm. A tunable 80 MHz titanium:sapphire femtosecond laser (710-920 nm) was used for the multiphoton tomography. We applied this high-resolution imaging tool to visualize A1-R bacteria targeting the Lewis lung cancer cells growing subcutaneously in nude mice. The tomographic images revealed that bacterially-infected cancer cells greatly expanded and burst and thereby lost viability. Similar results were seen in vitro using confocal microscopy. The bacteria targeted the tumor within minutes of tail-vein injection. Using mice in which the nestin-promoter drives GFP and in which blood vessels are labeled with GFP, the bacteria could be imaged in and out of the blood vessels. Collagen scaffolds within the tumor were imaged by second harmonic generation (SHG). The multiphoton tomographic system described here allows imaging of cancer cell killing by bacteria and can therefore be used to further understand its mechanism and optimization for clinical application.


Multiphoton multispectral fluorescence lifetime tomography for the evaluation of basal cell carcinomas

Patalay R, Talbot C, Alexandrov Y, Lenz MO, Kumar S, Warren S, Munro I, Neil MA, König K, French PM, Chu A, Stamp GW, Dunsby C.

PLoS One. 2012;7(9):e43460. doi: 10.1371


We present the first detailed study using multispectral multiphoton fluorescence lifetime imaging to differentiate basal cell carcinoma cells (BCCs) from normal keratinocytes. Images were acquired from 19 freshly excised BCCs and 27 samples of normal skin (in & ex vivo). Features from fluorescence lifetime images were used to discriminate BCCs with a sensitivity/specificity of 79%/93% respectively. A mosaic of BCC fluorescence lifetime images covering >1 mm(2) is also presented, demonstrating the potential for tumour margin delineation. Using 10,462 manually segmented cells from the image data, we quantify the cellular morphology and spectroscopic differences between BCCs and normal skin for the first time. Statistically significant increases were found in the fluorescence lifetimes of cells from BCCs in all spectral channels, ranging from 19.9% (425-515 nm spectral emission) to 39.8% (620-655 nm emission). A discriminant analysis based diagnostic algorithm allowed the fraction of cells classified as malignant to be calculated for each patient. This yielded a receiver operator characteristic area under the curve for the detection of BCC of 0.83. We have used both morphological and spectroscopic parameters to discriminate BCC from normal skin, and provide a comprehensive base for how this technique could be used for BCC assessment in clinical practice.


Multiphoton laser tomography and fluorescence lifetime imaging of basal cell carcinoma: morphologic features for non-invasive diagnostics

Seidenari S, Arginelli F, Dunsby C, French P, König K, Magnoni C, Manfredini M, Talbot C, Ponti G.

Exp Dermatol. 2012 Nov;21(11):831-6. doi: 10.1111/j.1600-0625.2012.01554.x. Epub 2012 Aug 7


Multiphoton laser tomography (MPT) combined with fluorescence lifetime imaging (FLIM) is a non-invasive imaging technique, which gives access to the cellular and extracellular morphology of the skin. The aim of our study was to assess the sensitivity and specificity of MPT/FLIM descriptors for basal cell carcinoma (BCC), to improve BCC diagnosis and the identification of tumor margins. In the preliminary study, FLIM images referring to 35 BCCs and 35 healthy skin samples were evaluated for the identification of morphologic descriptors characteristic of BCC. In the main study, the selected parameters were blindly evaluated on a test set comprising 63 BCCs, 63 healthy skin samples and 66 skin lesions. Moreover, FLIM values inside a region of interest were calculated on 98 healthy skin and 98 BCC samples. In the preliminary study, three epidermal descriptors and 7 BCC descriptors were identified. The specificity of the diagnostic criteria versus 'other lesions' was extremely high, indicating that the presence of at least one BCC descriptor makes the diagnosis of 'other lesion' extremely unlikely. FLIM values referring to BCC cells significantly differed from those of healthy skin. In this study, we identified morphological and numerical descriptors enabling the differentiation of BCC from other skin disorders and its distinction from healthy skin in ex vivo samples. In future, MPT/FLIM may be applied to skin lesions to provide direct clinical guidance before biopsy and histological examination and for the identification of tumor margins allowing a complete surgical removal.


Diagnosis of BCC by multiphoton laser tomography

Seidenari S, Arginelli F, Bassoli S, Cautela J, Cesinaro AM, Guanti M, Guardoli D, Magnoni C, Manfredini M, Ponti G, König K.

Skin Res Technol. 2013 Feb;19(1):e297-304. doi: 10.1111/j.1600-0846.2012.00643.x. Epub 2012 Jul 8.


BACKGROUND/PURPOSE:  Multiphoton Laser Tomography (MPT) is a non-linear optical technique that gives access to morphology and structure of both cells and extracellular matrix of the skin through the combination of autofluorescence imaging and second harmonic generation (SHG). The aim of this study was to identify MPT descriptors on ex vivo specimens of basal cell carcinoma (BCC) to assess the sensitivity and specificity of these criteria for the diagnosis of BCC and its differentiation from other skin tumours, inflammatory diseases and healthy skin. METHODS:  In the preliminary study, MPT images referring to 24 BCCs and 24 healthy skin samples were simultaneously evaluated by three observers for the identification of features characteristic of BCC. In the main study, the presence/absence of the descriptors identified in the preliminary study was blindly evaluated on a test set, comprising 66 BCCs, 66 healthy skin samples and 66 skin lesions, including 23 nevi, 8 melanomas, 17 skin tumours and other skin lesions by 3 independent observers. RESULTS:  In the preliminary study, three epidermal descriptors and six descriptors for BCC were identified. The latter included aligned elongated cells, double alignment of cells, cell nests with palisading and phantom islands. From the test set, 56 BCCs were correctly diagnosed, whereas in 10 cases the diagnosis was 'other lesions'. However, it was always possible to exclude the diagnosis of BCC in healthy skin and other lesion samples. Thus, overall sensitivity of the method was 84.85, whereas a specificity of 100% was observed with respect to both healthy skin and 'other lesions'. CONCLUSIONS:  This study describes new morphological descriptors of BCC enabling its characterization and its distinction from healthy skin and other skin lesions in ex vivo samples, and demonstrates for the first time that MPT represents a sensitive and specific technique for the diagnosis of BCC.


Three-dimensional multiphoton/optical coherence tomography for diagnostic applications in dermatology

Alex A, Weingast J, Weinigel M, Kellner-Höfer M, Nemecek R, Binder M, Pehamberger H, König K, Drexler W.

J Biophotonics. 2012 Jun 18. doi: 10.1002/jbio.201200085.


A preliminary clinical trial using state-of-the-art multiphoton tomography (MPT) and optical coherence tomography (OCT) for three-dimensional (3D) multimodal in vivo imaging of normal skin, nevi, scars and pathologic skin lesions has been conducted. MPT enabled visualization of sub-cellular details with axial and transverse resolutions of <2 μm and <0.5 μm, respectively, from a volume of 0.35 × 0.35 × 0.2 mm(3) at a frame rate of 0.14 Hz (512 × 512 pixels). State-of-the-art OCT, operating at a center wavelength of 1300 nm, was capable of acquiring 3D images depicting the layered architecture of skin with axial and transverse resolutions ∼8 μm and ∼20 μm, respectively, from a volume of 7 × 3.5 × 1.5 mm(3) at a frame rate of 46 Hz (1024 × 1024 pixels). This study demonstrates the clinical diagnostic potential of MPT/OCT for pre-screening relatively large areas of skin using 3D OCT to identify suspicious regions at microscopic level and subsequently using high resolution MPT to obtain zoomed in, sub-cellular level information of the respective regions (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).


Non-invasive evaluation of dermal elastosis by in vivo multiphoton tomography with autofluorescence lifetime measurements

Koehler MJ, Preller A, Elsner P, König K, Hipler UC, Kaatz M.

Exp Dermatol. 2012 Jan;21(1):48-51. doi: 10.1111/j.1600-0625.2011.01405.x.


The non-invasive differentiation of dermal elastic fibres from solar elastosis in vivo is of great interest in dermatologic research, especially for efficacy testing of anti-ageing products. To date, no studies on multiphoton excited fluorescence lifetime characteristics of human elastic fibres and solar elastosis are reported. The goal of the present work was the identification of differential criteria for elastic fibres and solar elastosis by the analysis of fluorescence decay curves acquired by time-correlated single photon counting in vivo multiphoton tomography. For this purpose, fluorescence lifetime measurements (FLIM) were performed with 47 volunteers of different age groups at sun-protected and sun-exposed localizations. Bi-exponential curve fitting was applied to the FLIM data, and characteristic differences between age groups and localizations were found in both relevant fit parameters describing the decay slope. The FLIM analyses have shown that dermal autofluorescence has different lifetimes depending on age and in part on localization.